Pseudomonas aeruginosa is the leading cause of ventilator-associated pneumonia (VAP), and current antibiotic treatment strategies exhibit failure rates as high as 18%, even when the organism is susceptible to the antibiotic being administered. The goal of this project is to address this critical medical need by validating targets in the type III secretion (T3SS) pathway that are susceptible to inhibition by small molecules and determining their roles in the T3SS host-pathogen interaction. T3SS is the major P. aeruginosa virulence determinant contributing to the establishment and dissemination of infections (e.g. VAP, bacteremia, urinary tract infections). It is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes, thereby weakening the host's innate immune defenses. The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients and markedly reduces survival in animal infection models. The strategy employed in this project is to use existing chemical probes to determine which components of the complex T3SS machine are susceptible to inhibition by small molecule compounds. Then, probes and strains carrying mutations in the probe targets will be used to define the roles of those vulnerable components in the host-pathogen interaction. Results will provide up to four well-validated, functionally annotated, druggable targets in the T3SS host-pathogen interaction. Four published T3SS inhibitors with unrelated chemical structures that are inhibitory to P. aeruginosa T3SS at non-cytotoxic concentrations have been selected as probes. Two of the probes are potent inhibitors of both T3SS-mediated secretion and translocation of effector toxins while the other two probes inhibit only secretion or translocation. Thus, these fou chemical probes likely inhibit at least three distinct targets or distinct regions within one or moe targets. In Aim 1, two parallel approaches will be used to identify the molecular targets of these four probes - (a) addition of photo-reactive and molecular handle moieties to permissible sites on the probes, application of photo-affinity probes to modify the target(s), recovery and identification of modified proteins; (b) application of molecular genetic tools to enrich for probe escape mutants followed by identification of the mutated gene(s) by deep sequencing. Finally, target identity will be confirmed by mutation analysis, and target gene mutant libraries will be prepared to facilitate understanding the role of each target in T3SS. In Aim 2, the probes and mutant libraries will be used to define the role of each probe target in the T3SS host-pathogen interaction. Effects of the probes and mutants on twelve distinct steps in the T3SS pathway within the broad categories of regulation, assembly, secretion, translocation, and cytotoxicity wil be used to dissect the roles of the targets in the T3SS machine and in host-pathogen interactions. Results will provide druggable, disease-relevant T3SS targets with characterized escape mutants for use in drug-discovery screening. Ideal targets will be prioritized as highly sensitive to rapid inhibition by probes and involved in critical roles in host-pathogen interaction.